PROJECT SUMMARY/ABSTRACT Adolescence represents a developmental phase where the skeleton is particularly susceptible to damage that can have long-term effects. Alcohol (ethanol) use is highly prevalent among adolescents, where 1 in 17 (~6%) of boys and girls aged 12-17 are estimated to have engaged in binge drinking. Ethanol is a bone toxicant known to generate oxidative stress by increasing intracellular reactive oxygen species (ROS). Additionally, ethanol decreases circulating levels of insulin-like growth factor-1 (IGF-1). Chondrocytes are specialized bone cells derived from the mesenchymal lineage that undergo a stepwise differentiation process ? consisting of proliferation, hypertrophy, and apoptosis ? to maintain the growth plate in mammalian long bones through the process of chondrogenesis. This process is influenced to a significant extent by IGF-1, which supports chondrocyte proliferation and hypertrophy and controls oxidative stress, and a balance of ROS signals generated by the NADPH oxidase enzymes Nox2 and Nox4. Interference with ROS regulation can impair normal growth plate homeostasis and longitudinal growth. Ethanol impairs growth plate maturation, although the precise mechanisms underlying this effect have not been elucidated in vivo. The studies proposed in this application are designed to uncover the mechanistic aspects of ethanol?s effects on the growth plate using an adolescent mouse model (C57Bl6 mice, aged 6 weeks) and chronic ethanol exposure (Lieber-DeCarli diet, 28% ethanol, 10 weeks). The overall hypothesis of these studies is that ethanol reduces IGF-1 signaling and induces multiple sources of intracellular ROS to suppress chondrocyte proliferation, disrupt growth plate formation, and impair longitudinal bone growth. Three specific aims will address complementary aspects of the systemic and intracellular toxicity of ethanol on chondrogenesis. Specific Aim 1 will use intravenous IGF-1 supplementation to determine the extent to which ethanol impairs IGF-1 signaling and suppresses growth plate maturation. Specific Aim 2 will use the mitochondria-targeted antioxidant mitoTEMPO to suppress mitochondrial ROS generation and determine their contribution to ethanol?s ROS-mediated effects. Specific Aim 3 will use separate Cre-lox genotypes that delete Nox2 and Nox4 specifically in chondrocytes to determine the role of each enzyme in ethanol-induced ROS, as well as each enzyme?s respective role in the distinct stages of chondrocyte differentiation. The responses to these experimental conditions will be determined by gene expression (qPCR, RNAscope hybridization), protein biology (western blotting, immunohistochemistry, ELISA), ROS generation (DMPO spin trapping), cellular morphology (histological staining), and overall bone structure. These studies will generate new insights into the role of hormonal factors and ROS in chondrocyte maturation and provide an integrated picture of ethanol?s mechanism of action on growth plate development during an important window of developmental susceptibility.